Human antibodies to human delta like ligand 4

ABSTRACT

The present invention provides methods of treating, ameliorating, or inhibiting tumor growth, cancer, or pathological angiogenesis by administering to a subject in need thereof a human antibody or fragment thereof that specifically binds to human delta-like ligand 4 (hDll4) and blocks hDll4 binding to a Notch receptor. The anti-hDll4 antibody or fragment thereof of the present invention have a high affinity with the KD of 500 pM or less, as measured by surface plasmon resonance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 16/658,521, filed on Oct. 21, 2019, which is a continuation of U.S. application Ser. No. 16/296,339, filed on Mar. 8, 2019, which is a continuation of U.S. patent application Ser. No. 16/035,990, filed on Jul. 16, 2018, which is a continuation of U.S. patent application Ser. No. 15/293,508, filed on Oct. 14, 2016, which is a continuation of U.S. patent application Ser. No. 14/729,218, filed on Jun. 3, 2015, which is a continuation of U.S. patent application Ser. No. 14/524,164, filed on Oct. 27, 2014, which is a continuation of U.S. patent application Ser. No. 14/176,523, filed on Feb. 10, 2014, which is a continuation of U.S. patent application Ser. No. 13/034,761, filed on Feb. 25, 2011, which is a divisional of U.S. non-provisional application Ser. No. 12/002,245, filed on Dec. 14, 2007, now U.S. Pat. No. 7,919,593, issued on Apr. 5, 2011, which claims the benefit under 35 U.S. C § 119 (e) of U.S. provisional application nos. 60/874,922 filed Dec. 14, 2006, 60/916,415 filed May 7, 2007, and 60/985,323 filed Nov. 5, 2007, all of which are herein specifically incorporated by reference in their entirety.

BACKGROUND

The Notch-signaling pathway is a system for cell-to-cell communication used by a wide range of eukaryotes for many biological processes, such as differentiation, proliferation, and homeostasis. Delta like 4 (Dl4) or delta-like ligand 4 (Dll4) (hereinafter “Dll4”) is a member of the Delta family of Notch ligands which exhibits highly selective expression by vascular endothelium (Shutter et al. (2000) Genes Develop. 14:1313-1318). Dll4 is a ligand for Notch receptors, including Notch1 and Notch 4. The nucleic acid and amino acid sequences for human Dll4 are shown in SEQ ID NO:1-2, respectively.

Methods to produce antibodies useful as human therapeutics include generation of chimeric antibodies and humanized antibodies (see, for example, U.S. Pat. No. 6,949,245). See, for example, WO 94/02602 (Abgenix) and U.S. Pat. No. 6,596,541 (Regeneron Pharmaceuticals), which publications are herein specifically incorporated by reference, describing methods of generating nonhuman transgenic mice capable of producing human antibodies.

Japanese patent application 2003/047470A2 (Asahi Kasei Kogyo) describes antibodies to the extracellular portion of human Notch ligand protein.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides human antibodies, preferably recombinant human antibodies, that specifically bind human delta-like ligand 4 (hDll4). These antibodies are characterized by binding to hDll4 with high affinity and by the ability to neutralize Dll4 activity. The antibodies of the invention are capable of blocking Dll4 binding to its Notch receptor(s), and thus inhibit signaling by Dll4. The antibodies can be full-length (for example, an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab′)₂ or scFv fragment), and may be modified to effect functionality, e.g., to eliminate residual effector functions (Glu which eliminates residual effector functions (Reddy et al. (2000) J. Immunol. 164:1925-1933).

In one embodiment, the antibody of the invention comprises a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NO:4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 397, 413, 429, 445, 461, 477, 493, 509, 525, 541, 557, 573, 589, 605, 621, 637, 653, 669, 685, 701, 717, 733, 749, 765, 781, 797, 813, 893, 897, 901, 905, 909, 913, 917, 921, 925, 935, 939, 943, and 947 or a substantially identical sequence thereof. In a preferred embodiment, the HCVR is the amino acid sequence of SEQ ID NO:429 or 901.

In one embodiment, the antibody of the invention comprises a light chain variable region (LCVR) selected from the group consisting of SEQ ID NO:12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 405, 421, 437, 453, 469, 485, 501, 517, 533, 549, 565, 581, 597, 613, 629, 645, 661, 677, 693, 709, 725, 741, 757, 773, 789, 805, 821, 895, 899, 903, 907, 911, 915, 919, 923, 927, 937, 941, 945, and 949 or a substantially identical sequence thereof. In a preferred embodiment, the LCVR is the amino acid sequence of SEQ ID NO:437 or 903.

In one embodiment, the antibody of the invention comprises a HCVR selected from the group consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 397, 413, 429, 445, 461, 477, 493, 509, 525, 541, 557, 573, 589, 605, 621, 637, 653, 669, 685, 701, 717, 733, 749, 765, 781, 797, 813, 893, 897, 901, 905, 909, 913, 917, 921, 925, 935, 939, 943, and 947 or a substantially identical sequence thereof, and a LCVR selected from the group consisting of SEQ ID NO:12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 405, 421, 437, 453, 469, 485, 501, 517, 533, 549, 565, 581, 597, 613, 629, 645, 661, 677, 693, 709, 725, 741, 757, 773, 789, 805, 821, 895, 899, 903, 907, 911, 915, 919, 923, 927, 937, 941, 945, and 949 or a substantially identical sequence thereof. In a preferred embodiment, the HCVR/LCVR are the amino acid sequence pairs SEQ ID NO:429/437 or 901/903.

In one embodiment, the invention features a human antibody or antibody fragment comprising a heavy chain complementary determining region 1 (CDR1) selected from the group consisting of SEQ ID NO:6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278 294, 310, 326, 342, 358, 374, 399, 415, 431, 447, 463, 479, 495, 511, 527, 543, 559, 575, 591, 607, 623, 639, 655, 671, 687, 703, 719, 735, 751, 767, 783, 799, 815, 831, 847, 863 and 879, or a substantially identical sequence thereof.

In one embodiment, the invention features a human antibody or antibody fragment comprising a heavy chain CDR2 selected from the group consisting of SEQ ID NO:8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 401, 417, 433, 449, 465, 481, 497, 513, 529, 545, 561, 577, 593, 609, 625, 641, 657, 673, 689, 705, 721, 737, 753, 769, 785, 801, 817, 833, 849, 865 and 881, or a substantially identical sequence thereof.

In one embodiment, the invention features a human antibody or antibody fragment comprising a heavy chain CDR3 selected from the group consisting of SEQ ID NO:10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 403, 419, 435, 451, 467, 483, 499, 515, 531, 547, 563, 579, 595, 611, 627, 643, 659, 675, 691, 707, 723, 739, 755, 771, 787, 803, 819, 835, 851, 867 and 883, or a substantially identical sequence thereof.

In one embodiment, the invention features a human antibody or antibody fragment comprising a heavy chain CDR1 selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278 294, 310, 326, 342, 358, 374, 399, 415, 431, 447, 463, 479, 495, 511, 527, 543, 559, 575, 591, 607, 623, 639, 655, 671, 687, 703, 711119, 735, 751, 767, 783, 799, 815, 831, 847, 863 and 879, or a substantially identical sequence thereof; a heavy chain CDR2 selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 401, 417, 433, 449, 465, 481, 497, 513, 529, 545, 561, 577, 593, 609, 625, 641, 657, 673, 689, 705, 721, 737, 753, 769, 785, 801, 817, 833, 849, 865 and 881, or a substantially identical sequence thereof; and a heavy chain CDR3 selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 403, 419, 435, 451, 467, 483, 499, 515, 531, 547, 563, 579, 595, 611, 627, 643, 659, 675, 691, 707, 723, 739, 755, 771, 787, 803, 819, 835, 851, 867 and 883, or a substantially identical sequence thereof. In a preferred embodiment, the antibody or antibody fragment comprises heavy chain CDR1, CDR2 and CDR3 selected from the group consisting of SEQ ID NO:431/433/435; 374/376/378; 783/785/787; and 799/801/803.

In one embodiment, the invention features a human antibody or antibody fragment comprising a light chain CDR1 selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, 407, 423, 439, 455, 471, 487, 503, 519, 535, 551, 567, 583, 599, 615, 631, 647, 663, 679, 695, 711, 727, 743, 759, 775, 791, 807, 823, 839, 855, 871 and 887, or a substantially identical sequence thereof.

In one embodiment, the invention features a human antibody or antibody fragment comprising a light chain CDR2 selected from the group consisting of SEQ ID NO:16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 409. 425, 441, 457, 473, 489, 505, 521, 537, 553, 569, 585, 601, 617, 633, 649, 665, 681, 697, 713, 729, 745, 761, 777, 793, 809, 825, 841, 857, 873 and 889, or a substantially identical sequence thereof.

In one embodiment, the invention features a human antibody or antibody fragment comprising a light chain CDR3 selected from the group consisting of SEQ ID NO:18, 34, 50, 66, 82, 98, 11, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555, 571, 587, 603, 619, 635, 651, 667, 683, 699, 715, 731, 747, 763, 779, 795, 811, 827, 843, 859, 875 and 891, or a substantially identical sequence thereof.

In one embodiment, the invention features a human antibody or antibody fragment comprising a light chain CDR1 selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, 407, 423, 439, 455, 471, 487, 503, 519, 535, 551, 567, 583, 599, 615, 631, 647, 663, 679, 695, 711, 727, 743, 759, 775, 791, 807, 823, 839, 855, 871 and 887, or a substantially identical sequence thereof; a light chain CDR2 selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 409. 425, 441, 457, 473, 489, 505, 521, 537, 553, 569, 585, 601, 617, 633, 649, 665, 681, 697, 713, 729, 745, 761, 777, 793, 809, 825, 841, 857, 873 and 889, or a substantially identical sequence thereof; and a light chain CDR3 selected from the group consisting of SEQ ID NO: 18, 34, 50, 66, 82, 98, 11, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555, 571, 587, 603, 619, 635, 651, 667, 683, 699, 715, 731, 747, 763, 779, 795, 811, 827, 843, 859, 875 and 891, or a substantially identical sequence thereof. In a preferred embodiment, the antibody or antibody fragment comprises the light chain CDR1, CDR2 and CDR3 selected from the group consisting of SEQ ID NO:439/441/443; 382/384/386; 791/793/795; and 807/809/811.

In a second aspect, the invention provides nucleic acid molecules encoding the antibodies, or antigen-binding portions, of the invention. Recombinant expression vectors carrying the antibody-encoding nucleic acids of the invention, and host cells into which such vectors have been introduced, are also encompassed by the invention, as are methods of making the antibodies of the invention by culturing the host cells of the invention.

In one embodiment, the antibody of the invention comprises a HCVR encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307, 323, 339, 355, 371, 396, 412, 428, 444, 460, 476, 492, 508, 524, 540, 556, 572, 588, 604, 620, 636, 652, 668, 684, 700, 716, 732, 748, 764, 780, 796, 812, 892, 896, 900, 904, 908, 912, 916, 920, 924, 934, 938, 942, and 946 or a substantially similar sequence having at least 95% homology thereof.

In one embodiment, the antibody of the invention comprises a LCVR encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, 580, 596, 612, 628, 644, 660, 676, 692, 708, 724, 740, 756, 772, 788, 804, 820, 894, 898, 902, 906, 910, 914, 918, 922, 926, 936, 940, 944, and 948 or a substantially similar sequence having at least 95% homology thereof.

In one embodiment, the antibody of the invention comprises a HCVR encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307, 323, 339, 355, 371, 396, 412, 428, 444, 460, 476, 492, 508, 524, 540, 556, 572, 588, 604, 620, 636, 652, 668, 684, 700, 716, 732, 748, 764, 780, 796, 812, 892, 896, 900, 904, 908, 912, 916, 920, 924, 934, 938, 942, and 946 or a substantially similar sequence having at least 95% homology thereof, and a LCVR encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, 580, 596, 612, 628, 644, 660, 676, 692, 708, 724, 740, 756, 772, 788, 804, 820, 894, 898, 902, 906, 910, 914, 918, 922, 926, 936, 940, 944, and 948 or a substantially similar sequence having at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibody fragment comprising a heavy chain CDR1 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:5, 21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, 309, 325, 341, 357, 373, 398, 414, 430, 446, 462, 478, 494, 510, 526, 542, 558, 574, 590, 606, 622, 638, 654, 670, 686, 702, 718, 734, 750, 766, 782, 798, 814, 830, 846, 862 and 878, or a substantially similar sequence having at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibody fragment comprising a heavy chain CDR2 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 100, 215, 231, 247, 263, 279, 295, 311, 327, 343, 359, 375, 400, 416, 432, 448, 464, 480, 496, 512, 528, 544, 560, 576, 592, 608, 624, 640, 656, 672, 688, 704, 720, 736, 752, 768, 784, 800, 816, 832, 848, 864 and 880, or a substantially similar sequence having at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibody fragment comprising a heavy chain CDR3 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345, 361, 377, 402, 418, 434, 450, 466, 482, 498, 514, 530, 546, 562, 578, 594, 610, 626, 642, 658, 674, 690, 706, 722, 738, 754, 770, 786, 802, 818, 834, 850, 866 and 882, or a substantially similar sequence having at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibody fragment comprising a heavy chain CDR1 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:5, 21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, 309, 325, 341, 357, 373, 398, 414, 430, 446, 462, 478, 494, 510, 526, 542, 558, 574, 590, 606, 622, 638, 654, 670, 686, 702, 718, 734, 750, 766, 782, 798, 814, 830, 846, 862 and 878, or a substantially similar sequence having at least 95% homology thereof; a heavy chain CDR2 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 100, 215, 231, 247, 263, 279, 295, 311, 327, 343, 359, 375, 400, 416, 432, 448, 464, 480, 496, 512, 528, 544, 560, 576, 592, 608, 624, 640, 656, 672, 688, 704, 720, 736, 752, 768, 784, 800, 816, 832, 848, 864 and 880, or a substantially similar sequence having at least 95% homology thereof; and a heavy chain CDR3 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345, 361, 377, 402, 418, 434, 450, 466, 482, 498, 514, 530, 546, 562, 578, 594, 610, 626, 642, 658, 674, 690, 706, 722, 738, 754, 770, 786, 802, 818, 834, 850, 866 and 882, or a substantially similar sequence having at least 95% homology thereof. In a preferred embodiment, the antibody or antibody fragment comprises heavy chain CDR1, CDR2 and CDR3 encoded b a nucleic acid sequence selected from the group consisting of SEQ ID NO:430/432/434; 373/375/377; 782/784/786; and 798/800/802.

In one embodiment, the invention features a human antibody or antibody fragment comprising a light chain CDR1 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, 301, 317, 333, 349, 365, 381, 406, 422, 438, 454, 470, 486, 502, 518, 534, 550, 566, 582, 598, 614, 630, 646, 662, 678, 694, 710, 726, 742, 758, 774, 790, 806, 822, 838, 854, 870 and 886, or a substantially similar sequence having at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibody fragment comprising a light chain CDR2 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, 303, 319, 335, 351, 367, 383, 408, 424, 440, 456, 472, 488, 504, 520, 536, 552, 568, 584, 600, 616, 632, 648, 664, 680, 696, 712, 728, 744, 760, 776, 792, 808, 824, 840, 856, 872, and 888, or a substantially similar sequence having at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibody fragment comprising a light chain CDR3 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337, 353, 369, 385, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554, 570, 586, 602, 618, 634, 650, 666, 682, 698, 714, 730, 746, 762, 778, 794, 810, 826, 842, 858, 874 and 890, or a substantially similar sequence having at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibody fragment comprising a light chain CDR1 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, 301, 317, 333, 349, 365, 381, 406, 422, 438, 454, 470, 486, 502, 518, 534, 550, 566, 582, 598, 614, 630, 646, 662, 678, 694, 710, 726, 742, 758, 774, 790, 806, 822, 835, 851, 867 and 883, or a substantially similar sequence having at least 95% homology thereof; a light chain CDR2 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, 303, 319, 335, 351, 367, 383, 408, 424, 440, 456, 472, 488, 504, 520, 536, 552, 568, 584, 600, 616, 632, 648, 664, 680, 696, 712, 728, 744, 760, 776, 792, 808, 824, 840, 856, 872, and 888, or a substantially similar sequence having at least 95% homology thereof; and a light chain CDR3 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337, 353, 369, 385, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554, 570, 586, 602, 618, 634, 650, 666, 682, 698, 714, 730, 746, 762, 778, 794, 810, 826, 842, 858, 874 and 890, or a substantially similar sequence having at least 95% homology thereof. In a preferred embodiment, the antibody or antibody fragment comprises the light chain CDR1, CDR2 and CDR3 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:438/440/442; 381/383/385; 790/792/794; and 806/808/810.

In a third aspect, the invention features an isolated antibody or antibody fragment that specifically binds hDll4, comprising a CDR 1, 2 and 3 selected from the group consisting of (a) a heavy chain CDR1 region comprising an amino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:928), wherein X¹ is Gly; X² is Phe or Tyr; X³ is Thr; X⁴ is Phe; X⁵ is Ser, Thr or Asn; X⁶ is Ser, Asn or Tyr; X⁷ is Tyr or Phe; and X⁸ is Gly or Ala; (b) a heavy chain CDR2 region comprising an amino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:929), wherein X¹ is Ile or Leu; X² is Trp or Ser; X³ is Tyr, Ala or Gly; X⁴ is Asp, Ser or Tyr; X⁵ is Gly or Asp; X⁶ is Ser, Gly, Thr or Val; X⁷ is Asn or Asp; and X⁸ is Lys or Arg; (c) a heavy chain CDR3 region comprising an amino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³-X¹⁴-X¹⁵-X¹⁶ (SEQ ID NO:930), wherein X¹ is Ala or Ser; X² is Arg or Lys; X³ is Asp or Tyr; X⁴ is Ser, Gly or His; X⁵ is Asp, Ala or Trp; X⁶ is Asn, or Phe; X⁷ is Tyr, Arg or Lys; X⁸ is His or Ser; X⁹ is Gly or Trp; X¹⁰ is Tyr or Phe; X¹¹ is Glu or Asp; X¹² is Gly, His or Pro; X¹³ is Tyr, Trp or absent; X¹⁴ is Phe or absent; X¹⁵ is Asp or absent; and X¹⁶ is Pro or absent.

In a preferred embodiment, the antibody or antibody fragment comprises heavy chain CDR 1, 2 and 3 selected from the group consisting of (a) a heavy chain CDR1 region comprising an amino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:928), wherein X¹ is Gly; X² is Phe; X³ is Thr; X⁴ is Phe; X⁵ is Ser or Asn; X⁶ is Ser or Asn; X⁷ is Tyr or Phe; and X⁸ is Gly or Ala; (b) a heavy chain CDR2 region comprising an amino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:929), wherein X¹ is Ile or Leu; X² is Trp or Ser; X³ is Tyr or Gly; X⁴ is Asp or Ser; X⁵ is Gly; X⁶ is Ser, Thr or Val; X⁷ is Asn or Asp; and X⁸ is Lys or Arg; (c) a heavy chain CDR3 region comprising an amino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³-X¹⁴-X¹⁵-X¹⁶ (SEQ ID NO:930), wherein X¹ is Ala or Ser; X² is Arg or Lys; X³ is Asp; X⁴ is Gly or His; X⁵ is Asp or Ala; X⁶ is Phe; X⁷ is Tyr or Arg; X⁸ is Ser; X⁹ is Gly; X¹⁰ is Tyr; X¹¹ is Glu; X¹² is Gly or His; X¹³ is Tyr or Trp; X¹⁴ is Phe or absent; X¹⁵ is Asp or absent; and X¹⁶ is Pro or absent.

In a further embodiment, the isolated antibody or antibody fragment further comprises (d) a light chain CDR1 region comprising an amino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:931), wherein X¹ is Gln; X² is Ser; X³ is Val; X⁴ is Arg, Ser or Thr; X⁵ is Ser or Gly; X⁶ is Ser or Tyr; and X⁷ is Tyr or absent; (e) a light chain CDR2 region comprising an amino acid sequence of the formula X¹-X²-X³ (SEQ ID NO:932), wherein X¹ is Gly or Asp; X² is Ala or Thr; and X³ is Ser; and (f) a light chain CDR3 region comprising an amino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ (SEQ ID NO:933), wherein X¹ is Gln; X² is Gln or His; X³ is Tyr, Arg or Ser; X⁴ is Gly, Ser or Ala; X⁵ is Ser, Asn or Phe; X⁶ is Trp or Ser; X⁷ is Pro; X⁸ is Trp, Pro or Arg; and X⁹ is Thr.

In a preferred embodiment, the isolated antibody or antibody fragment further comprises (d) a light chain CDR1 region comprising an amino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:931), wherein X¹ is Gln; X² is Ser; X³ is Val; X⁴ is Arg or Ser; X⁵ is Ser; X⁶ is Ser or Tyr; and X⁷ is Tyr or absent; (e) a light chain CDR2 region comprising an amino acid sequence of the formula X¹-X²-X³ (SEQ ID NO:932), wherein X¹ is Gly or Asp; X² is Ala or Thr; and X³ is Ser; and (f) a light chain CDR3 region comprising an amino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ (SEQ ID NO:933), wherein X¹ is Gln; X² is Gln or His; X³ is Tyr or Arg; X⁴ is Gly or Ser; X⁵ is Ser or Asn; X⁶ is Trp or Ser; X⁷ is Pro; X⁸ is Pro or Arg; and X⁹ is Thr.

In a fourth aspect, the invention features a fully human antibody or antibody fragment which binds hDll4 with an IC₅₀ of less than about 10 nM, as measured in in vitro assay or ELISA-based Dll4 blocking assay (described below). In a preferred embodiment, the antibody of the invention exhibits an IC₅₀ of about 500 pM or less. In an even more preferred embodiment, the antibody of the invention exhibits an IC₅₀ of about 100 pM or less.

In one embodiment, the invention provides a fully human monoclonal antibody which specifically binds and inhibits human Dll4 and exhibits an IC₅₀ of less than or equal to about 150 pM, 100 pM, 75 pM, or 50 pM, as measured by Notch-inducible luciferase bioassay with hDll4-Fc. As shown in the experimental section below, the anti-hDll4 antibodies of the invention do not cross-react with closely related delta proteins, such as hDll1 and hDll3.

In one embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, that binds hDll4 with a K_(D) of less than about 500 pM, preferably less than about 300 pM, even more preferably less than about 100 pM, less than about 50 pM, less than about 10 pM, as determined by surface plasmon resonance (BIACORE™), for example, using dimeric hDll4 (Table 2).

The invention encompasses anti-hDll4 antibodies having a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of a galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).

The invention includes anti-hDll4 antibodies which bind specific epitopes of hDll4 and are capable of blocking the biological activity of hDll4. The extracellular domain of Dll4 is composed of an N-terminal domain, a Delta/Serrate/Lag-2 (DSL) domain, and a tandem of eight epidermal growth factor (EGF)-like repeats. Generally, the EGF domains are recognized as occurring at about amino acid residues 218-251 (domain 1), 252-282 (domain 2), 284-322 (domain 3), 324-360 (domain 4), and 362-400 (domain 5), with the DSL domain at about amino acid residues 173-217 and the N-terminal domain at about amino acid residues 27-172 of hDll4 (SEQ ID NO:2).

In one embodiment, a blocking antibody of the invention binds within amino acids residues 27 to 524 of SEQ ID NO:2. In a more specific embodiment, a blocking antibody of the invention binds an epitope within the N-terminus-DSL domains 27-217 of SEQ ID NO:2; in an even more specific embodiment, the blocking antibody binds an epitope within about amino acid residues 27-172 (N-terminal domain) or 173-217 (DSL domain). In another embodiment, a blocking antibody of the invention binds the EGF-2 epitope within about amino acids residues 252-282 of SEQ ID NO:2.

In a fifth aspect, the invention features a composition comprising a recombinant human anti-human Dll4 antibody and an acceptable carrier. Further included in the invention are vectors and host cells comprising vectors which contain nucleic acid molecules encoding the human anti-hDll4 antibody of the invention, as well as methods of producing these novel antibodies, comprising growing a host cell comprising nucleic acid encoding the anti-hDll4 antibody of the invention or an antibody fragment, under conditions permitting production of the protein and recovering the protein so produced.

In a sixth aspect, the invention features methods for inhibiting hDll4 activity using an antibody, or antigen-binding portion thereof, of the invention. In one embodiment, the method comprises contacting hDll4 with the instant antibody or antigen-binding portion thereof, such that hDll4 is inhibited from binding to Notch receptor, for example Notch-1. In another embodiment, the method comprises administering an antibody or antibody fragment of the invention, to a human subject suffering from a disorder which is ameliorated by inhibition of Dll4 activity. The disorder treated is a disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition or reduction of Dll4 activity, for example, pathological vascularization associated with tumor angiogenesis and cancer, immunodeficiency diseases, transplant rejection, or inflammation; and neurodegenerative conditions, e.g., associated with prion disease.

Other objects and advantages will become apparent from a review of the ensuing detailed description.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

Definitions

“Delta-like ligand 4”, “Dll4”, “hDll4” are used interchangeably to refer to the protein encoded by the nucleic acid sequence of SEQ ID NO:1 and the protein having the amino acid sequence of SEQ ID NO:2.

The term “antibody”, as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementary determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term “high affinity” antibody refers to those antibodies having a binding affinity to hDll4 of at least 10⁻⁸ M; preferably 10⁻⁹ M; even more preferably 10⁻¹⁰ M, as measured by surface plasmon resonance, e.g., BIACORE™ or solution-affinity ELISA.

By the term “slow off rate” or “Koff” is meant an antibody that dissociates from hDll4 with a rate constant of 1×10⁻³ s⁻¹ or less, preferably 1×10⁻⁴ s⁻¹ or less, as determined by surface plasmon resonance, e.g., BIACORE™.

A “neutralizing” or “blocking” antibody, is intended to refer to an antibody whose binding to Dll4 results in inhibition of the biological activity of Dll4. This inhibition of the biological activity of Dll4 can be assessed by measuring one or more indicators of Dll4 biological activity. These indicators of Dll4 biological activity can be assessed by one or more of several standard in vitro or in vivo assays known in the art (see examples below). Preferably, the ability of an antibody to neutralize Dll4 activity is assessed by inhibition of Dll4 binding to a Notch receptor.

The term “antigen-binding portion” of an antibody (or simply “antibody portion” or “antibody fragment”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hDll4). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature 241:544-546), which consists of a VH domain; and (vi) an isolated CDR. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad Sci. USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂ fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

An “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds hDll4 is substantially free of antibodies that specifically bind antigens other than hDll4). An isolated antibody that specifically binds hDll4 may, however, have cross-reactivity to other antigens, such as hDll4 molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction.

The term “epitope” includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In preferred embodiments, an antibody is said to specifically bind an antigen when the equilibrium dissociation constant is less than or equal to 10⁻⁸ M, more preferably when the equilibrium dissociation constant is less than or equal to 10⁻⁹ M, and most preferably when the dissociation constant is less than or equal to 10⁻¹⁰ M.

A protein or polypeptide is “substantially pure,” “substantially homogeneous” or “substantially purified” when at least about 60 to 75% of a sample exhibits a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

The term “polypeptide analog or variant” as used herein refers to a polypeptide that is comprised of a segment of at least 25 amino acids that has substantial identity to a portion of an amino acid sequence and that has at least one of the following properties: (1) specific binding to hDll4 under suitable binding conditions, or (2) ability to block Dll4 binding to a Notch receptor. Typically, polypeptide analogs or variants comprise a conservative amino acid substitution (or insertion or deletion) with respect to the naturally-occurring sequence. Analogs typically are at least 20 amino acids long, preferably at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.

Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various mutations of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton 1984 W. H. Freeman and Company, New York; Introduction to Protein Structure (Branden & Tooze, eds., 1991, Garland Publishing, NY); and Thornton et at. 1991 Nature 354:105, which are each incorporated herein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry as drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics” (see, for example, Fauchere (1986) J. Adv. Drug Res. 15:29; and Evans et al. (1987) J. Med. Chem. 30:1229, which are incorporated herein by reference. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may also be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo et al. (1992) Ann. Rev. Biochem. 61:387, incorporated herein by reference), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

The term “percent sequence identity” in the context of nucleic acid sequences refers to the residues in two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides or more, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestf it, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA, which includes, e.g., the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (1990) Methods Enzymol. 183:63-98 and (2000) Methods Mol. Biol. 132:185-219, each herein incorporated by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference.

A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. Generally, the art uses the terms “percent sequence identity”, “percent sequence similarity” and “percent sequence homology” interchangeably. In this application, these terms shall have the same meaning with respect to nucleic acid sequences.

The term “substantial similarity”, or “substantial sequence similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, preferably at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

As applied to polypeptides, the term “substantial identity” or “substantially identical” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80% sequence identity, preferably at least 90% or 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; and 6) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403 410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389 402, each of which is herein incorporated by reference.

The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences.

Preparation of Human Antibodies

Methods for generating human antibodies include, for example, VELOCIMMUNE® (Regeneron Pharmaceuticals), XENOMOUSE™ technology (Abgenix), the “minilocus” approach, and phage display. The VELOCIMMUNE® technology (U.S. Pat. No. 6,596,541) encompasses a method of generating a high specificity fully human antibody to a select antigen. This technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody. In specific embodiment, the cell is a CHO cell.

The XENOMOUSE™ technology (Green et al. (1994) Nature Genetics 7:13-21) generates a mouse having both human variable and constant regions from both the heavy chain and kappa light chain loci. In an alternative approach, others have utilized a ‘minilocus” approach in which an exogenous Ig locus is mimicked through inclusion of individual genes from the Ig locus (see, for example, U.S. Pat. No. 5,545,807). The DNA encoding the variable regions can be isolated with or without being operably linked to the DNA encoding the human heavy and light chain constant region.

Other methods of generating human antibodies, including isolation from a human donor, are known. See, for example, U.S. Pat. No. 6,787,637, herein specifically incorporated by reference in its entirety.

Antibodies may be therapeutically useful in blocking a ligand-receptor interaction or inhibiting receptor component interaction, rather than by killing cells through fixation of complement and participation in CDC. The constant region of an antibody is important in the ability of an antibody to fix complement and participate in CDC or direct cell killing through antibody-dependent cellular cytoxicity (ADCC). Thus, the isotype of an antibody may be selected on the basis of the desirability for the antibody to fix complement.

Human immunoglobulins can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via inter-heavy chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a single light and heavy chain. These forms have been difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. In fact, a single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. 1993 Molecular Immunology 30:105) to levels typically observed using a human IgG1 hinge. The instant invention encompasses antibodies having one or more mutations in the hinge, CH2 or CH3 region which may be desirable, for example, in production to improve the yield, or modulate effector functions.

Antibodies of the invention are preferably prepared with the use of VELOCIMMUNE® technology. A transgenic mouse in which the endogenous immunoglobulin heavy and light chain variable regions are replaced with the corresponding human variable regions is challenged with the antigen of interest, and lymphatic cells (such as B-cells) recovered from the mice that express antibodies. The lymphatic cells may be fused with a myeloid-type cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimeric antibodies may be isolated directly from antigen-specific lymphocytes. In various embodiments, the transgenic mouse comprises 12 functional human variable heavy chain genes and 11 functional human variable kappa light chain genes; 25 to 30 human variable heavy chain genes and from 18 to 20 human variable kappa light chain genes; 43 to 48 human variable heavy chain genes and 20 to 22 human variable kappa light chain genes; or about 80 human variable heavy chain genes and about 40 human variable kappa light chain genes.

In general, the antibodies of the instant invention possess very high affinities, typically possessing K_(D) of from about 10⁻⁹ through about 10⁻¹¹ M, when measured by binding to antigen either immobilized on solid phase or in solution phase. The mouse constant regions are replaced with desired human constant regions to generate the fully human antibodies of the invention, for example wild-type or modified IgG1 or IgG4 (for example, SEQ ID NO:950, 951, or 952). While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.

Cancer, infectious diseases, autoimmunity, immunodeficiency, transplants, inflammation, injury and degenerative conditions can be treated by modulation of the immune system. In cases of disease due to inappropriate function or hyperactivity of the immune system, such as autoimmunity or inflammation, can be ameliorated through inhibition of immune cell function or reduction of immune cell numbers. This can be accomplished by blockade of positive signals or stimulation of negative signals on immune cell populations critical to the disease process, such as T, B or NK cells, neutrophils, macrophages, antigen presenting cells, mast cells or other cell types. Overactivity can also be inhibited through elimination of various immune cell populations by stimulation of apoptosis, targeting of specific surface receptors with depleting antibodies or antibody-drug conjugates, or the blockade or alteration of the differentiation of immune cell lineages or specific cell types. Inefficient or reduced immune function can cause or exacerbate disorders such as cancer, infectious disease, and other immunodeficiencies. Hypoactivity of the immune system can be improved through activation of immune cells by stimulation of positive signals by crosslinking or agonistic antibodies or blockade of negative signals. Immune cell populations can be increased by stimulation of development of some or all immune cell lineages, prevention of apoptosis, or elimination of inhibitory signals. In a specific application, the antibodies of the invention are useful for treatment, inhibition or amelioration of a condition or disease such as, for example, cancer, immunodeficiency, transplant rejection, or inflammation.

Epitope Mapping and Related Technologies

To screen for antibodies which bind to a particular epitope (e.g., those which block binding of IgE to its high affinity receptor), a routine cross-blocking assay such as that described in Harlow and Lane (1990) supra can be performed. Other methods include alanine scanning mutants, peptide blots (Reineke (2004) Methods Mol Biol 248:443-63) (herein specifically incorporated by reference in its entirety), or peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9: 487-496) (herein specifically incorporated by reference in its entirety).

The term “epitope” refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (U.S. patent Publication No. 2004/0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the hDll4 antibodies of the invention into groups of antibodies binding different epitopes.

Agents useful for altering the structure of the immobilized antigen are enzymes, such as, for example proteolytic enzymes, for example, trypsin, endoproteinase Glu-C, endoproteinase Asp-N, chymotrypsin, etc. Agents useful for altering the structure of the immobilized antigen may also be chemical agents, such as, succinimidyl esters and their derivatives, primary amine-containing compounds, hydrazines and carbohydrazines, free amino acids, etc.

The antigen protein may be immobilized on either biosensor chip surfaces or polystyrene beads. The latter can be processed with, for example, an assay such as multiplex LUMINEX™ detection assay (Luminex Corp., Austin, TX). Because of the capacity of LUMINEX™ to handle multiplex analysis with up to 100 different types of beads, LUMINEX™ provides almost unlimited antigen surfaces with various modifications, resulting in improved resolution in antibody epitope profiling over a biosensor assay.

Therapeutic Administration and Formulations

The administration of therapeutic entities in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA). These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol. 52:238-311 and the citations therein for additional information related to excipients and carriers well known to pharmaceutical chemists.

EXAMPLES Example 1. Generation of Human Antibodies to Human Dll4

Mice may be immunized by any method known in the art (see, for example, Harlow and Lane supra). In one embodiment, hDll4 antigen is administered directly to VELOCIMMUNE® mice comprising DNA loci encoding human Ig heavy chain variable regions and kappa light chain variable regions, with an adjuvant to stimulate the immune response. Such an adjuvant includes complete and incomplete Freund's adjuvant, MPL+TDM adjuvant system (Sigma), or RIBI (muramyl dipeptides) (see O'Hagan 2000 Vaccine Adjuvant, by Human Press, Totawa, NJ). The antibody immune response is monitored by standard antigen specific immunoassay. When a desired immune response is achieved, antibody-expressing B cells were harvested and fused with mouse myeloma cells to preserve their viability, forming hybridoma cell lines. Such hybridoma cell lines are screened and selected to identify cell lines that produce antigen-specific antibodies using assays as described below.

Alternatively, antigen-specific hybridoma cells may be isolated by flow cytometry. Briefly, after fusion to myeloma cells, pooled hybridoma cells were grown for 10 days in HAT medium. The cells were then harvested and stained with biotin-labeled Dll4 at 2 mg/ml for one hour, followed by addition of phycoerythrin-streptavidin. The fluorescence-labeled cells were sorted by flow cytometry (single cell per well into 96 well plates containing hybridoma growth medium), cultured for 8-10 days, and conditioned media screened for the presence of functionally desirable monoclonal antibodies, as described below.

Anti-hDll4 antibodies generated via direct isolation of splenocytes. Antigen-specific antibodies may also be isolated directly from antigen-immunized B cells without fusion to myeloma cells, as described in U.S. Patent Publication 2007/0280945A1, herein specifically incorporated by reference in its entirety. Stable recombinant antibody-expressing CHO cell lines were established from the isolated proper recombinants.

Example 2. Antigen Binding Affinity Determination

Equilibrium dissociation constants (K_(D) values) for antigen binding to the selected antibodies described above were determined by surface kinetics on a real-time biosensor surface plasmon resonance assay (BIACORE™ 2000). The antibody was captured on a goat anti-mouse IgG polyclonal antibody surface created through direct chemical coupling to a BIACORE™ chip to form a captured antibody surface. Varying concentrations of monomeric hDll4 or dimeric hDll4-hFc were injected over the captured antibody surfaces, and antigen-antibody binding and dissociation monitored in real time. Kinetic analysis was performed to calculate K_(D), dissociation rate constants, and half-life of antigen/antibody complex dissociation (Table 1). A similar method was applied to measure single B cell-derived monoclonal antibodies modified to contain a human IgG constant domain. Antibodies were presented by goat anti-hFc polyclonal antibody reagent (Jackson Immuno Research Lab) immobilized on BIACORE™ chip, and exposed to either dimeric Dll4-mFc or monomeric Dll4 protein (Table 2).

Antibody-antigen binding affinity may also be assessed using an ELISA based solution competition assay. Briefly, on a 96-well microtiter plate, antibodies (purified proteins or in conditioned medium) were premixed with serial dilutions of antigen protein (monomeric or dimeric) ranging from 0 to 10 μg/ml with a constant concentration of antibody. After a 2 hr incubation of antigen with antibody, the solutions were transferred to a microtiter plate precoated with antigen for measurement of free antibody (MAXISORB™, VWR, West Chester, PA). The plate was coated with 1 μg/ml hDll4-hFc protein in PBS solution overnight at 4° C. and nonspecific binding sites blocked with BSA for 2 hrs. After a 1 hr incubation following transfer, the plate was washed and the plate-bound antibodies were detected with an HRP-conjugated goat anti-mouse IgG polyclonal antibody reagent (Jackson Immuno Laboratory) and developed using colorimetric substrates (OPTEIA™; BD Biosciences Pharmingen, San Diego, CA). The enzymatic reaction was stopped with 1 M phosphoric acid, optical absorptions at 450 nm were recorded and the data were analyzed using a sigmoidal dose-response model and an IC₅₀ values were reported (Table 1).

TABLE 1 K_(D) DII4 K_(D) DII4-Fc IC₅₀ DII4-Fc Antibody (nM) (nM) (nM) 13B6 2.79 0.188 0.06 15E10 0.55 0.023 0.58 22G12 1.29 0.076 0.03 24C8 0.52 0.047 0.01 VAV 2H4-19 1.51 0.611 0.10 VAV 4H10-9 13.70 0.662 0.30 VAV 7B9-9 0.88 0.021 0.27 VAW 10E4-9 89.00 0.468 0.06 VAW 10G11-2 31.30 1.430 1.66 VAW 1C6-1 45.80 0.092 0.25 VAW 1G2-4 83.80 0.035 0.40 VAW 1H2-2 67.00 0.148 0.30 VAW 2H3-2 0.30 0.150 0.26 VAW 3A7-2 1.64 0.162 0.02 VAW 3A9-5 NA 2.510 16.00 VAW 3F12-8 8.12 0.648 0.07 VAW 6B8-12 0.89 0.060 0.43 VAW 6C6-2 91.70 0.092 0.50 VAW 6G12-10 3.74 0.527 0.19 VAW 7C10-11 17.10 0.853 0.28 VAW 8A10-14 1.41 0.648 0.08 VAW 8G1-12 6.09 8.300 8.60 VAW 9B11-2 62.20 0.048 0.00 VAW 9F12-6 16.00 1.350 0.02 VAW 9G10-1 56.10 0.555 0.10

TABLE 2 K_(D) DII4 K_(D) DII4-Fc Antibody (nM) (nM) 314266-06F12-B7 2.17 0.075 318518-01A04-D5 0.237 0.244 318518-01A10-D8 0.399 0.018 318518-01B09-C3 0.833 0.180 318518-01B11-D4 0.382 0.088 318518-01E07-H2 0.165 0.238 318518-01G04-F3 0.501 0.107 318518-01G05-B5 1.06 0.196 318518-02A07-B3 0.208 0.148 318518-02B06-E2 2.15 0.193 318518-02B08-F7 N/A N/A 318518-02C04-D1 0.478 0.331 318518-02F05-D10 1.28 0.035 318518-02G03-F2 1.31 0.042 318518-02G04-B11 0.813 0.048 318518-02G08-F11 N/A N/A 318518-03A03-B2 0.136 0.124 318518-03C10-F2 1.18 0.131 318518-03D04-B5 0.904 0.136 318518-03D07-G11 3.74 0.163 318518-03F04-A6 0.501 0.088 318518-03F06-A3 0.556 0.037 318518-03H03-F3 8.89 0.084 318518-14A06-E7 4.54 0.282 318518-14A07-C4 0.235 0.035 318518-14D08-G1 0.541 0.046 318518-14H08-A2 6.67 0.128 318518-1H08-E9 0.225 0.050

Example 3. Inhibition of Dll4 and Notch Interaction

The ability of the antibodies to block Dll4 binding to Notch was evaluated with an ELISA-based immunoassay. Briefly, Notch-hFc recombinant protein was coated on a 96-well plate in PBS buffer overnight at 4° C. at 1 mg/ml, and the nonspecific binding sites were blocked with BSA. This plate was used to measure free biotin-Dll4-hFc from antibody titration sample solutions. To make the antibody titration samples, a constant amount of biotin-Dll4-hFc at 25 pM was pre-mixed with varied amounts of antibody, either in crude hybridoma condition medium or as purified antibody protein, ranging from 0 to ˜50 nM in serial dilutions, followed by 2 hr incubation at room temperature to allow antibody-antigen binding to reach equilibrium. The equilibrated sample solutions were then transferred to the Notch-hFc coated plates for the measurement of free biotin-Dll4-hFc. After 1 hour binding, the plate was washed and bound biotin-Dll4-hFc was detected using HRP conjugated streptavidin (Poly HRP streptavidin, Pierce Endogen), and developed using TMB substrate (BD Pharmigen). Data was analyzed using Graph Pad Prism software and IC₅₀ values were determined as the amount of antibody required to achieve 50% reduction of biotin-Dll4-hFc bound to the plate-coated Notch-Fc (Table 3) (*conditioned media).

TABLE 3 Antibody IC₅₀ (nM) VAV 2H4-19 0.01 VAW 3A7-2 0.017 VAW 9G10-1 0.019 VAW 10E4-9 0.032 VAW 8A10-11 0.04 VAW 9F12-6 0.059 VAW 3F12-8 0.066 VAW 1C6-1 0.086 VAW 1G2-4 0.11 VAW 6C6-2 0.119 VAV 7B9-4 0.123 VAW 1H2-2 0.154 VAW 2H3-2 0.168 VAW 6B8-12 0.255 VAW 6G12-10 0.257 VAW 7C10-11 0.273 VAV 4H10-9 0.599 VAW 10G11-2 0.931 VAW 3A9-5 3.8 VAW 8G1-12 10.7 VAW 9B11-2 0.069 15E10* 0.04 22G12 0.10 13B6 0.11 24C8 0.031 314266-06F12-B7 0.07 318518-01A04-D5 0.11 318518-01A10-D8 0.05 318518-01B09-C3 0.03 318518-01B11-D4 0.03 318518-01E07-H2 1.17 318518-01G04-F3 0.02 318518-01G05-B5 1.08 318518-02A07-B3 0.03 318518-02B06-E2 0.09 318518-02B08-F7 N/A 318518-02C04-D1 0.60 318518-02F05-D10 0.16 318518-02G03-F2 0.07 318518-02G04-B11 1.09 318518-02G08-F11 N/A 318518-03A03-B2 0.57 318518-03C10-F2 0.12 318518-03D04-B5 0.04 318518-03D07-G11 0.45 318518-03F04-A6 0.01 318518-03F06-A3 0.02 318518-03H03-F3 0.17 318518-14A06-E7 0.04 318518-14A07-C4 0.02 318518-14D08-G1 0.14 318518-14H08-A2 0.25 318518-1H08-E9 0.03

The ability of selected purified anti-hDll4 antibodies to block Dll4 binding to Notch was also evaluated with the ELISA-based immunoassay described above, modified by replacing 25 pM of biotin-Dll4-hFc with 30 pM of biotin-Dll4-hFc, and reducing antibody-antigen incubation duration from 2 hrs to 1 hr. For convenience, antibody 318518-01A10-D8 was renamed “REGN281” (HCVR/LCVR SEQ ID NOs:429/437 and hIgG1 SEQ ID NO:950). Derived antibodies tested included REGN421 (HCVR/LCVR SEQ ID NO:901/903, hIgG1 SEQ ID NO:950); and REGN422 (HCVR/LCVR SEQ ID NO:901/903, with modified hIgG4 SEQ ID NO:952). Results are shown in Table 4.

TABLE4 Antibody IC₅₀ (nM) REGN281 0.042 REGN421 0.045 REGN422 0.039

Ability of antibody to neutralize Dll4 mediated cellular function was also tested in vitro using Dll4 expressing human umbilical vein endothelial cells (HUVEC). Inhibition of Notch mediated hHes1 and EphB2 gene expression in HUVEC with the derived antibodies was monitored as follows: low passage HUVEC were cultured in MCDB-131 media (Vec Technologies). One day prior to analysis, HUVEC cells were seeded at a density of 2×10⁵ cells/well in 24-well plates in 1 ml total media volume. The test antibody or other inhibitor was added directly to the individual sample wells in triplicate followed by 5-hour culture at 37° C. At the end of the culture period, media was removed and total RNA was isolated using QIAZOL™ and the RNEASY™ lipid tissue kit (Qiagen). mRNA level quantification was performed by the use of PCR and the fluorogenic 5′ nuclease assay (TAQMAN® assay, Applied Biosystems). For each sample, cDNA was synthesized from 1-2 mg of total RNA. cDNA generated from an equivalent amount of starting RNA (typically 25 ng) was loaded in triplicate on ABI PRISM™ optical reaction plates. For each RNA sample a “no RT” control was also run in which no reverse transcriptase was added to allow for subtraction of any potential genomic DNA contributions to the signal. 2× Mastermix (TAQMAN® 2×PCR Mastermix; ABI) was added to each reaction to a final concentration of 1×. Additionally, TAQMAN® probe and primers for the gene of interest were added to each reaction. Each primer was used at a final concentration of 900 nM and the probe was added at a final concentration of 200 nM. Human genomic DNA was used as a standard. The assays were performed under standard TAQMAN® conditions on a ABI 7900HT instrument. Levels of Hes1 and EphrinB2 were measured and normalized to an endogenous control gene (cyclophilin) (Table 5). Probes and primers: human Hes1 probe (SEQ ID NO:387); Oligos: hHes1-869F (SEQ ID NO:388); hHes1-940R (SEQ ID NO:389), human ephrinB2 probe: hEphB2-773T (SEQ ID NO:390); Oligos: hEphB2-752F (SEQ ID NO:391) hEphB2-812R (SEQ ID NO:392); human cyclophilin: probe: hCyclophilin-343T (SEQ ID NO:393); Oligos: hCyclophilin-323F (SEQ ID NO:394); hCyclophilin-389R (SEQ ID NO:395).

TABLE 5 EphrinB2 Hes1 Expression Expression Antibody IC₅₀ (nM) IC₅₀ (nM) 22G12 0.379 0.381 15E10 2.56 4.49 VAW 3A7-2 0.409 0.533 314266-06F12-B7 0.239 0.405 318518-01A10-D8 0.305 0.329 318518-01G04-F3 0.088 0.172 318518-01H08-E9 0.413 0.548 318518-02A07-B3 0.398 0.128 318518-03F04-A6 0.158 0.115 318518-03F06-A3 0.304 0.692 318518-014A07-C4 0.175 0.312 318518-014D08-G1 0.510 0.568 hDII4-hFc 0.843 0.974

HUVEC Proliferation Assay. The ability of antibody to block Dll4 mediated growth inhibition of Human umbilical vein endothelial cells (HUVEC) cells was tested in an in vitro cell proliferation assay. Low passage HUVEC cells were obtained and cultured in MCDB-131 media (Vec Technologies). One day prior to analysis 12-well tissue culture plates were coated with hDll4-hFc in PBS at 4° C. overnight (0.2 μg/ml; 0.5 ml PBS/well). Plates were washed 1× with PBS and HUVEC cells were seeded at a density of 4×10³ cells/well in 1.0 ml total media volume. Immediately following addition of cells anti-hDll4 antibodies were added in 0.5 ml total volume over a range of concentrations to generate an inhibition curve. Cells were grown for 96-hours at 37° C. Cell number was quantitated using the CCK-8 reagent (Dojindo). All assays were run in triplicate. (Table 6, NB, not blocking).

TABLE 6 Antibody IC₅₀ (nM) 15E10 0.284 VAW9B11-2 1.868 VAW8D8-12 NB 13B6 5.01  VAW2H4-19 NB VAW3A7-2 0.198 VAW8A10-14 0.214 22G12 0.888 318518-06F12-B7 2.067 318518-01A10-D8 0.096 318518-01G04-F3 0.106 318518-01H08-E9 0.188 318518-02A07-B3 0.200 318518-03F04-A6 0.184 318518-03F06-A3 0.188 318518-014A07-C4 0.159 318518-014D08-G1 0.165

Notch-Inducible Luciferase Assay. A bioassay was developed to determine the ability of selected purified antibodies to neutralize Dll4-mediated cellular function in vitro using an engineered HEK293 cell line (ATCC® (American Type Culture Collection)) that constitutively expresses human Notch 1 and contains a Notch-responsive promoter driving luciferase. Inhibition of Notch-inducible luciferase activity was determined as follows: 1 day prior to assay, each well of an opaque 96 well tissue culture plate was coated with 100 μl of either 1 nM or 1.5 nM hDll4-hFc in PBS overnight at 4° C. Cells were seeded onto the coated plates at 2×10⁴ cells/well in media. Purified antibody protein, in serial dilutions starting from 2 nM in cell media, was incubated with the cells at 37° C. for 24 hrs. Luciferase activity was determined by adding an equal well volume of STEADY-GLO® Substrate (Promega) (Table 7).

TABLE 7 IC₅₀ (pM) Antibody 1 nM hDII4-hFc 1.5 nM hDII4-hFc REGN281 50.5 78.7 REGN421 54.4 87.3 REGN422 88.2 131.1

Example 4. Inhibition of Notch1 Cleavage

The ability of selected anti-hDll4 antibodies to inhibit Notch1 cleavage was tested by examination of total cleaved Notch1 protein by SDS-Page/Western blotting. Low passage HUVEC cells were cultured as described above. One day prior to analysis 6-well plates were coated with hDll4-hFc in PBS at 4° C. overnight (0.2 μg/ml; 1.0 ml PBS/well). Plates were washed 1× with PBS and HUVEC cells were seeded at 7.5×10⁵ cells/well in 2.0 ml total media volume. Immediately following cell seeding, anti-hDll4 antibody was added to each well at 10 nM final concentration. Cells were grown for 24 hours at 37° C. following which whole cell extracts were prepared and analyzed by SDS-PAGE. Levels of cleaved Notch1 were determined using an anti-cleaved Notch1 (Val1744) antibody (Cell Signaling) and standard western blotting techniques. The anti-hDll4 antibodies were able to entirely block the Notch1 cleavage induced by plate coated hDll4-hFc (data not shown).

Example 5. ADCC and CDC Assays

Antibody-dependent cell-mediated cytotoxicity (ADCC) induced by two test antibodies (REGN421, REGN422) was assessed using a panel of eight target cell lines with varying hDll4 expression levels. The eight target cell lines were (1) HUVECs; (2) HUVECs stimulated with 10 nM VEGF for 24 hours; (3) Colo205; (4) engineered C6 rat glioma cells expressing eGFP; (5) engineered C6 rat glioma cells expressing hDll4; (6) engineered HT1080 cells expressing eGFP; (7) engineered HT1080 cells expressing hDll4; and (8) HT29. Human Dll4 or eGFP was integrated into the C6 cell or HT1080 genome through retroviral transfection. Briefly, cells from each target cell line (10,000 cells/well in 50 μl) were first mixed with an equal volume of serially diluted REGN421 or REGN422, resulting in a final antibody concentration ranging from 0.169 pM to 10 nM, and incubated for 10 min at room temperature in a 96-well plate format (control=wells without antibody). Separately, human peripheral blood mononuclear cells (PBMCs, effector cells) were prepared following a conventional Ficoll-Hypaque gradient centrifugation enrichment procedure. Approximately 300,000 PBMCs were added to each mixture of antibody and target cells to give a final ratio of effector to target cells of approximately 30:1. The 96-well plates were then incubated for 4 h at 37° C., 5% CO₂ followed by centrifugation at 250×g. Supernatants were harvested and assayed for lactate dehydrogenase (LDH) activity using the CYTOTOX 96® Non-Radioactive Cytotoxicity Assay system (Promega). Results are shown in Table 8. REGN421-induced dose dependent cell lysis was only observed in C6 cells expressing hDll4 (col. 5), which exhibited the highest hDll4 expression among all cell lines (as determined by immunoprecipitation/Western blot and flow cytometry). The maximum cell cytotoxicity in the C6-hDll4 cell line ranged from 20% to 60%. No REGN421-induced cell lysis was observed in the remaining seven target cell lines. REGN422 did not induce cell lysis in any of the target cell lines.

TABLE 8 % Maximum Cytotoxicity Ab 1 2 3 4 5 6 7 8 REGN421 0 0 0 0 20-60 0 0 0 REGN422 0 0 0 0 0 0 0 0

Complement-dependent cytotoxicity (CDC) induced by REGN421 was assessed using the same panel of cells lines described above. Briefly, cells from each of the target cell lines (50,000 cells/well in 50 μl) were first mixed with an equal volume of serially diluted REGN421, resulting in a final antibody concentration ranging from 0.169 pM to 10 nM, and incubated for 10 min at room temperature in a 96-well plate format. Normal human serum, with complement components (Quidel Corp., San Diego, CA) was added to each well to give a final serum concentration of 5%. The plates were then incubated at 37° C., 5% CO₂ for 2 hours followed by addition of CELLTITER-BLUE® reagent (Promega) (controls=wells without antibody and wells with antibody but no serum). The plates were incubated overnight and cell survival (CDC levels) assayed. As a positive control, Daudi cells were treated with rituximab. REGN421 exhibited no CDC toward any of the target cell lines tested (data not shown).

Example 6. Epitope Mapping and Specificity

In order to determine epitope binding specificity, a series of seven chimeric Dll4 proteins were generated in which specific human Dll4 domains were substituted into a mouse Dll4 protein as follows: #1 contained the human N-terminal and DSL domains (S27-Q218); #2 contained human N-terminal, DSL and EGF-1 domains (S27-N252); #3 contained human N-terminal, DSL, EGF-1 and EGF-2 domains (S27-Q283); #4 contained human N-terminal, DSL, EGF-1, EGF-2, EGF-3, EGF-4 and EGF-5; #5 contained human N-terminal domain (S27-R172); #6 contained human DSL domain (V173-Q218); and #7 contained human EGF-2 domain (E252-D282). The chimeric proteins were fused to a mouse IgG2a-Fc fragment and expressed in CHO-K1 cell. The conditioned media were harvested and protein expression confirmed by western blot.

Binding specificity of test antibodies to hDll4, mDll4, and chimeric proteins #1, #2, #3, and #4 were tested as follows: purified antibodies 22G12, VAW3A7-2, and 15E10 were amine coupled between 5000-6000 RU on CM5 chip. Conditioned media from CHO K1 cells containing the chimeric Dll4 proteins, hDll4-mFc, and mDll4-mFc were injected sequentially followed by surface regeneration over antibody-coupled surfaces. A blank amine coupled flowcell surface was used as a control for nonspecific binding of the conditioned media. Results are summarized in Table 9. 22G12 bound an epitope between S27-Q218 of hDll4; VAW3A7-2 bound an epitope between Q283-E400 hDll4; and 15E10 bound an epitope between E252-D282 of hDll4.

TABLE 9 Chimeric Proteins Antibody hDII4-mFc mDII4-mFc #1 #2 #3 #4 22G12 + − + + + + VAW3A7-2 + − − − − + 15E10 + − − − + +

Binding specificity of purified test mAb to hDll4, mDll4 and the chimeric proteins (described above) was determined (REGN279=314266-6F12-B7; REGN287=318518-1G04-F3; REGN289=318518-1H08-E9; REGN290=318518-2A07-B3; REGN306=318518-3F06-A3). Briefly, each Dll4 protein was captured (70-130 RU) on goat anti-mouse IgG antibody surfaces, followed by injection of test mAb at a concentration of 100 μg/ml. An antibody that bound mDll4-mFc was used as a positive control (positive control=6C10). The results (Table 10) show that REGN279 bound an epitope between S27-Q218 of hDll4; REGN287 bound between Q283-E400 of hDll4; REGN289, REGN290 and REGN306 bound between S27-E400 of hDll4.

TABLE 10 Chimeric human-mouse DII4 Fusion Proteins Antibody hDII4-mFc mDII4-mFc #1 #2 #3 #4 #5 #6 #7 REGN279 + − + + + + + − − REGN287 + − − − − + − − − REGN289 + − + + + + − + − REGN290 + − + + + + − + − REGN306 + − + + + + − + − Control + + + + + + + + +

Further epitope binding specificity determinations were conducted as described above with the following purified test antibodies: REGN281=318518-1A10-D8; REGN305=318518-3F04-A6; REGN309=318518-14A07-C4; REGN310=318518-14D08-G1; REGN421, and REGN422. Briefly, each of the Dll4 proteins was captured (240-470 RU) on goat anti-mouse IgG antibody surfaces, followed by injection of test antibody at a concentration of 100 μg/ml (Table 11).

TABLE 11 Chimeric human-mouse DII4 Fusion Proteins Antibody hDII4-mFc mDII4-mFc #1 #2 #3 #4 #5 #6 #7 REGN281 + − + + + + + + − REGN305 + − − − − + − − − REGN309 + − + + + − + + − REGN310 + − − − + + − + + REGN421 + − + + + + + + − REGN422 + − + + + + + + −

Western blot analysis. Binding specificity of selected antibodies to chimeric, mouse and human Dll4 was determined by Western blot. Briefly, hDll4-mFc (200 ng per lane), mDll4-mFc (200 ng per lane), and chimeric proteins #1-#7 (approximate 150 ng per lane) were subjected to electrophoresis on duplicate SDS-PAGE gels using non-reducing sample buffer. Each gel was then transferred to a PVDF membrane. Blots were first exposed to REGN421 at 0.2 μg/ml and then to HRP-conjugated anti-hIgG antibody (Pierce). Control blots were exposed to HRP-conjugated anti-mFc antibody (Pierce). Results: REGN421 recognized hDll4-mFc and chimeric proteins containing the human N-terminal domain (#5), a human DSL domain (#6), or both (#1, #2, #3, and #4). REGN421 did not recognize a chimeric protein containing a human EGF-2 domain (#7).

Protease digestion analysis. Binding between REGN281 and hDll4 was further assessed by protective protease digestion and liquid chromatography/mass spectrometry (LC/MS) using an HPLC1100 (Agilent) and LCQ Classical Ion Trap Mass Spectrometer (Thermo). Briefly, a mixture of hDll4 and REGN281, in a molar ratio of 1:5, or hDll4 alone, was incubated with protease overnight at either 25° C. (for GluC protease) or 37° C. (for trypsin). Each of the resulting proteolytic digest mixtures was then subjected to LC/MS. Unique peptide peaks present in proteolytic digests performed in the absence of REGN281, which either diminished or disappeared in proteolytic digests performed in the presence of REGN281, indicate potential REGN281 binding sites on hDll4 that were protected from protease digestion by the binding of REGN281 to hDll4. These unique peptide peaks were analyzed by mass spectrometry. The observed mass, predicted mass, and the N-terminal sequences of the peptides are shown in Table 12.

TABLE 12 Observed Predicted hDII4 Peptide Peak Mass Mass (SEQ ID NO: 2) Protease Domain G1 521 521.5 Phe37-Glu40 GluC N-terminal G2 1362.8 1363.4 Ala121-Glu132 GluC N-terminal T1 758 760.8 Pro49-Arg55 Trypsin N-terminal T2 587.1 587.2 Tyr169-Arg172 Trypsin N-terminal T3 1607.4 1607.6 Val173-Arg186 Trypsin DSL T4 1399 1400.7 Gly42-Arg55 Trypsin N-terminal T5 569.2 569.3 Thr56-Arg59 Trypsin N-terminal T7 2615 2613.4 Ile143-Arg166 Trypsin N-terminal T8 1807.2 1806.9 Ser27-Arg41 Trypsin N-terminal

Example 7. Binding Affinity of Purified Antibodies to Human and Monkey Dll4

The binding affinities of selected purified antibodies to hDll4, M. facscicularis Dll4 (mfDll4, SEQ IN NO:956), and M. mulatta Dll4 (mmDll4, SEQ ID NO:957) monomers were determined using a BIACORE™ 2000 & 3000. Goat anti-hFc polyclonal antibody reagent immobilized on a BIACORE™ chip was used to present REGN281, REGN421, and REGN422. Varying concentrations of each proteins, hDll4 (from 12.5 nM to 100 nM), mfDll4 (from 3.13. nM to 100 nM), or mmDll4 (from 12.5 nM to 100 nM) were used as analyte, and injected over the antibody surfaces. Antigen-antibody binding and dissociation of bound complex were monitored in real time (Table 13).

γTABLE 13 ka (M−1s−1) kd (s−1) K_(D) (nM) hDII4 mfDII4 mmDII4 hDII4 mfDII4 mmDII4 hDII4 mfDII4 mmDII4 REGN281 1.56 × 10⁵ 6.64 × 10⁴ 9.27 × 10⁴ 2.30 × 10⁻⁵ 2.04 × 10⁻⁵ 3.05 × 10⁻⁵ 0.148 0.307 0.329 REGN421 1.63 × 10⁵ 7.28 × 10⁴ 9.70 × 10⁴ 2.17 × 10⁻⁵ 2.02 × 10⁻⁵ 3.23 × 10⁻⁵ 0.133 0.278 0.333 REGN422 1.64 × 10⁵ 8.01 × 10⁴ 9.27 × 10⁴ 2.36 × 10⁻⁵ 2.88 × 10⁻⁵ 3.41 × 10⁻⁵ 0.144 0.360 0.375

The binding affinities of anti-hDll4 antibodies toward hDll4 and mmDll4 dimers were also determined using a BIACORE™ 2000 and the method describe above, except that hDll4 was replaced with hDll4-mFc (from 3.13 nM to 100 nM), or mmDll4 with mmDll4-mFc (from 0.78 nM to 25 nM) as analyte (Table 14).

TABLE 14 ka (M−1s−1) kd (s−1) K_(D) (nM) hDII4-mFc mmDII4-mFc hDII4-mFc mmDII4-mFc hDII4-mFc mmDII4-mFc REGN281 3.02 × 10⁵ 3.16 × 10⁵ 4.96 × 10⁻⁶ 4.64 × 10⁻⁶ 0.0163 0.0147 REGN421 3.43 × 10⁵ 3.35 × 10⁵ 4.70 × 10⁻⁶ 3.80 × 10⁻⁶ 0.0137 0.013 REGN422 3.46 × 10⁵ 4.23 × 10⁵ 4.60 × 10⁻⁶ 4.15 × 10⁻⁶ 0.0133 0.0098

Example 8. Cross-Reactivity of Antibodies with hDll1, hDll3, mDll4, or mfDll4

Cross-reactivity of the antibodies to human delta-like ligand1 (SEQ ID NO:953) and human delta-like ligand 3 (SEQ ID NO:954) proteins was determined. REGN281, REGN421 and REGN422 were presented by a goat anti-human kappa (hK) polyclonal antibody reagent (Southern Biotech) immobilized on a BIACORE™ chip, and either hDll4-hFc or hDll1-hFc protein at 100 μg/ml were used as analyte injected over the antibody surfaces. All three anti-hDll4 antibodies only bound to hDll4-hFc, and did not bind hDll1-hFc.

An alternative BIACORE™ format was used to assess cross-reactivity between anti-hDll4 antibody and either hDll1-hFc or hDll3-hFc. Briefly, ligands hDll4-hFc, hDll1-hFc, and hDll3-hFc were each covalently linked to a CM-5 chip, through amine coupling, to an RU range of about 8,000 to 10,000. REGN421, at 300 μg/ml, was injected over the surface of each chip. REGN421 bound only to hDll4-hFc; no binding was observed to either hDll1-hFc or hDll3-hFc. The same result was observed for REGN422 instead of REGN421.

OCTET™-based binding assays were employed to determine the binding between selected purified anti-hDll4 antibodies and hDll4-hFc, hDll3-hFc, hDll1-hFc, mfDll4-mmh, or mDll4-mFc. Briefly, strepavidin high binding FA biosensors (ForteBio, Inc., Menlo Park, CA) were first incubated with biotin-anti-hK at 5 μg/ml for 10 min at 30° C., to achieve saturation. Biotin-anti-hK-bound biosensors were then incubated with antibodies REGN281, REGN421 or REGN422, at 20 μg/ml for 10 min at 30° C., to achieve saturation. The antibody-bound biosensors were then incubated with either hDll4-hFc or hDll3-hFc, hDll1-hFc, or mDll4-mFc, at 200 nM, or mfDll4-mmh at 100 nM, for 10 min at 30° C. Changes in the thickness of the biological layer after each incubation were measured. Human Dll4-hFc and mfDll4-mmh bound to anti-hDll4 antibody-bound biosensors, whereas hDll3-hFc, hDll1-hFc, and mDll4-mFc did not bind to anti-hDll4 antibody-bound biosensors.

Example 9. Effect of Anti-hDll4 Antibody on Tumor Growth

The effect of REGN421 on tumor growth was evaluated on tumors implanted in Severe Combined Immunodeficiency (SCID) mice expressing a humanized Dll4 protein (SCID×hDll4). Briefly, the humanized Dll4 mouse was made by replacing the entire extracellular domain of the mouse Dll4 gene with the corresponding extracellular region of the human Dll4 gene (7 kb) in embryonic stem (ES) cells. Homozygous hDll4 mice were generated and bred into SCID background. Each mouse was then implanted subcutaneously (SC) with 2.5×10⁶ human HT1080 tumor cells. After the tumors were established in the mice (˜100-150 mm³ 18 days after implantation), mice were measured and treated with hFc, hDll4-Fc or REGN421. A total of 7 mice were divided into three groups. The first group (n=3) was treated subcutaneously with hFc at 25 mg/kg; the second group (n=1) was treated with hDll4-Fc at 25 mg/kg; and the third group (n=3) was treated with REGN421 at 10 mg/kg. The treatments were repeated every 48 hours starting at day 18. In vivo tumor measurements were obtained three days before the initial treatment (day 15), on the same day of each treatment (days 18, 20, and 22), and on day 25. Tumor size was calculated using the formula l×w²/2. Results are shown in Table 15. On day 25, mice were euthanized and each tumor was removed and measured ex vivo and calculated (length×width×depth) (Table 16).

In addition, a group of SCID mice expressing endogenous mDll4 (n=2) was implanted with tumor cells and treated with hDll4-Fc (25 mg/kg) following the same dosing schedule.

TABLE 15 Tumor Size (mm³) Mouse Treatment Day 15 Day 18 Day 20 Day 22 Day 25 SCID hDII4-hFc 162.0 232.8 320.0 336.0 253.1 SCID hDII4-hFc 22.5 117.0 117.0 108.0 68.8 SCIDxhDII4 hDII4-hFc 288.0 320.0 352.0 446.0 320.0 SCIDxhDII4 hFc 162.0 288.0 320.0 500.0 550.0 SCIDxhDII4 hFc 162.0 220.5 352.0 662.0 661.5 SCIDxhDII4 hFc 93.8 135.0 179.6 352.0 726.0 SCIDxhDII4 REGN421 144.0 245.0 320.0 162.0 144.0 SCIDxhDII4 REGN421 87.5 162.0 153.0 225.0 135.0 SCIDxhDII4 REGN421 144.0 196.0 272.0 162.0 152.5

TABLE 16 Tumor Size Mouse Treatment (mm³) SCID hDII4-hFc 308.0 SCID hDII4-hFc 105.0 SCID × hDII4 hDII4-hFc 480.0 SCID × hDII4 hFc 924.0 SCID × hDII4 hFc 1020.0 SCID × hDII4 hFc 792.0 SCID × hDII4 REGN421 168.0 SCID × hDII4 REGN421 84.0 SCID × hDII4 REGN421 189.0 

We claim:
 1. A method of treating, ameliorating, or inhibiting tumor growth or cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or fragment thereof that specifically binds human delta-like ligand 4 (Dll4) and blocks Dll4-Notch signal pathways, such that the tumor growth or cancer is treated, ameliorated, or inhibited, wherein the tumor growth or cancer expresses Dll4, and wherein the antibody or fragment thereof comprises a heavy chain variable region (HCVR) comprising heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOS:431, 433 and 435, respectively, and a light chain variable region (LCVR) comprising light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOS:439, 441 and 443, respectively.
 2. The method of claim 1, wherein the antibody or fragment thereof comprises a HCVR comprising the amino acid sequence of SEQ ID NO:429 or
 901. 3. The method of claim 1, wherein the antibody or fragment thereof comprises a LCVR comprising the amino acid sequence of SEQ ID NO:437 or
 903. 4. The method of claim 1, wherein the antibody or fragment thereof comprises a HCVR comprising the amino acid sequence of SEQ ID NO:429 and a LCVR comprising the amino acid sequence of SEQ ID NO:437.
 5. The method of claim 1, wherein the antibody or fragment thereof comprises a HCVR comprising the amino acid sequence of SEQ ID NO:901 and a LCVR comprising the amino acid sequence of
 903. 